The long-term objective of this project is to develop new antibiotics to combat pathogenic bacteria that may be used as agents of bioterrorism. This proposal focuses on developing small molecule inhibitors of siderophore biosynthesis in two bacteria that are NIAID Priority Pathogens for biodefense research: Yersinia pestis, the causative agent of plague, and Yersinia enterocolitica, a gastroenteritic water- and foodborne pathogen. Siderophores are iron-chelating natural products that mediate bacterial uptake of iron from the mammalian host, a process that is critical for bacterial virulence and growth. In the two bacteria above, the required first steps in siderophore biosynthesis are catalyzed by salicylate adenylation enzymes. Thus, inhibitors of these enzymes should block siderophore biosynthesis and, hence, bacterial iron uptake, growth, and virulence. We recently developed the first such inhibitor, called salicyl-AMS, which has potent activity against salicylate adenylation enzymes in biochemical assays (Ki = 0.35-1.08 nM). Salicyl-AMS also inhibits bacterial siderophore biosynthesis and growth under iron-limiting conditions, albeit with more modest potency (IC50 = 51.2[unreadable]M). To evaluate this new antibiotic strategy fully in cellular and animal infection models, inhibitors with improved cellular activity must be developed. In view of its potent biochemical activity, we hypothesize that the cellular activity of salicyl-AMS is being constrained by one or more pharmacological factors: permeability, efflux, stability, and/or specificity. We will use a collaborative, multidisciplinary approach to evaluate this hypothesis and to develop analogs with increased cellular activity through three integrated specific aims: (1) Synthesize salicyl-AMS analogs designed to provide improved permeability, efflux resistance, and stability;(2) Synthesize analogs designed to provide improved specificity based on an inhibitor binding model;(3) Test salicyl-AMS and its analogs in a panel of assays to evaluate the pharmacological factors that affect cellular activity and to identify optimized inhibitors. This project will set the stage for future evaluation of the most promising inhibitors in animal infection models to validate salicylate adenylation enzymes and siderophore biosynthesis as new antibiotic targets to combat bioterrorism. Public Health Relevance Statement: This project aims to develop new antibiotics to treat plague, yersiniosis, and other infectious diseases that are important in biodefense and global public health.